s2fieldsofstudy: Biology Psychology

text:
Neuroplastic changes in c‐Fos, ΔFosB, BDNF, trkB, and Arc expression in the hippocampus of male Roman rats: differential effects of sexual activity

Abstract Sexual activity causes differential changes in the expression of markers of neural activation (c‐Fos and ΔFosB) and neural plasticity (Arc and BDNF/trkB), as determined either by Western Blot (BDNF, trkB, Arc, and ΔFosB) or immunohistochemistry (BDNF, trkB, Arc, and c‐Fos), in the hippocampus of male Roman high (RHA) and low avoidance (RLA) rats, two psychogenetically selected rat lines that display marked differences in sexual behavior (RHA rats exhibit higher sexual motivation and better copulatory performance than RLA rats). Both methods showed (with some differences) that sexual activity modifies the expression levels of these markers in the hippocampus of Roman rats depending on: (i) the level of sexual experience, that is, changes were usually more evident in sexually naïve than in experienced rats; (ii) the hippocampal partition, that is, BDNF and Arc increased in the dorsal but tended to decrease in the ventral hippocampus; (iii) the marker considered, that is, in sexually experienced animals BDNF, c‐Fos, and Arc levels were similar to those of controls, while ΔFosB levels increased; and (iv) the rat line, that is, changes were usually larger in RHA than RLA rats. These findings resemble those of early studies in RHA and RLA rats showing that sexual activity influences the expression of these markers in the nucleus accumbens, medial prefrontal cortex, and ventral tegmental area, and show for the first time that also in the hippocampus sexual activity induces neural activation and plasticity, events that occur mainly during the first phase of the acquisition of sexual experience and depend on the genotypic/phenotypic characteristics of the animals.

| INTRODUCTION
Sexual behavior is a highly instinctually and hormonally driven stereotyped behavior; however, with many other behaviors, it has also to be learned and is modulated by experience (see Pfaus et al., 2012 and references therein). Indeed, the sexual experience causes several changes in sexual behavior making copulation more efficient and increasing sexual motivation. Accordingly, (i) sexually experienced male rats show an increased preference for receptive female rats, and better performances in classical copulatory tests than sexually unexperienced (naïve) male rats and (ii) sexual experience increases the incentive value of sexual stimuli and associated cues (Everitt, 1990;Pfaus & Everitt, 1995). In addition, the sexual experience causes also neurochemical and morphological changes in several brain areas, the most studied being the ventral tegmental area (VTA)nucleus accumbens (Acb) pathway (Beloate et al., 2016;Fiorino et al., 1997;Pfaus & Everitt, 1995) and hypothalamic areas such as the medial preoptic area (MPOA) (Dominguez & Hull, 2005) and the paraventricular nucleus of the hypothalamus (PVN) (Argiolas & Melis, 2005;Melis & Argiolas, 2021a). These changes are mediated by molecular mechanisms that play a key role not only in the behavioral modifications in sexual performance induced by sexual experience but also in the rewarding and reinforcing effects of sexual activity (Pitchers et al., 2013). Indeed, sexual experience induces changes in the pattern of the increase in dopamine release that usually occurs in the Acb, MPOA, and PVN of male rats during sexual activity and copulation (Fiorino et al., 1997;Hull et al., 1995;Meisel & Mullins, 2006;Melis et al., 2003). The increases in dopamine release occurring in the above brain areas are associated with an increased expression of the transcriptional factor c-Fos (a marker of neuronal activation) and of ΔFosB, a truncated form of c-Fos, in the same areas and the VTA (Biały & Kaczmarek, 1996;Bradley & Meisel, 2001;Hedges et al., 2009;Lumley & Hull, 1999;McHenry et al., 2016;Nishitani et al., 2004;Nutsch et al., 2016;Pitchers, Frohmader, et al., 2010;Veening & Coolen, 1998;Wallace et al., 2008;Witt & Insel, 1994), and MAPK phosphorylation has been observed in limbic areas (i.e., MPOA, olfactory bulb, and amygdala) of male mice after copulation (Taziaux et al., 2011). Furthermore, changes in the number of dendrites and dendritic spines in the Acb of sexually experienced male rats have been observed (Pitchers, Balfour, et al., 2010).
Recent studies have shown that the changes in the expression of the above markers of neural activation and plasticity are differentially influenced by the genotypic/phenotypic characteristics of the animals used to study the sexual behavior. Among these, the most studied are the Roman high (RHA) and low avoidance (RLA) rats (Giorgi et al., 2007 and the membrane dopamine transporter knockout (DAT-KO) rats and their wild type (WT) and heterozygous (HET) counterparts . RHA and RLA rats are two rat lines originally selected for their extremely divergent responses in the active avoidance task. They show different, often opposite behavioral traits (RHA rats are active copers, impulsive and novelty seekers, and prone to intake classical drugs of abuse, while RLA rats are reactive copers that show neophobia, anxiety, hyperemotional responses like freezing to novel situations, and are prone to develop depressive-like symptoms) (Giorgi et al., 2007. DAT-KO rats with their WT and HET counterparts differ in their dopaminergic tone, the first having a very high tone due to the DAT absence, the second a normal tone, and the third showing a dopaminergic tone intermediate between DAT-KO and WT rats . Important for this work, both Roman and DAT-KO rat lines display significant differences in sexual behavior, RHA rats and DAT-KO rats exhibiting higher sexual motivation and higher levels of sexual activity than RLA rats  and HET/WT rats (Sanna et al., 2020), respectively; besides, these differences in sexual behavior appear to be secondary to the higher tone of the mesocorticolimbic dopaminergic system present not only in DAT-KO versus HET/WT rats (Sanna et al., 2020) but also in RHA versus RLA rats (Sanna et al., 2015;Sanna, Bratzu, Piludu, et al., 2017). Moreover, the above mentioned behavioral and neurochemical differences occur concomitantly with significant differences not only in the expression of c-Fos and ΔFosB, but also of the brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase B (trkB) and the activityregulated cytoskeleton-associated protein (Arc) in the mesocorticolimbic system (i.e., VTA, Acb, and mPFC) after the exposition to, and direct sexual interaction with a sexually receptive female rat, not only in RHA versus RLA rats  but also in DAT-KO versus HET/WT rats (Sanna et al., 2020). These differences were bigger in sexually naïve rats (i.e., after their first sexual experience) but still present, although reduced, in sexually experienced rats (i.e., after five copulatory tests). Taken together with the differences found in the mesocorticolimbic dopamine tone, the differences in the expression of the above markers occurring in the limbic system between RHA and RLA rats, and DAT-KO rats and HET/WT rats, led to suggest that they could be responsible, at least in part, of the differences observed in sexual behavior and, more in general, in motivated behavior that characterize the two Roman rat lines  and DAT-KO rats (Cinque et al., 2018;Sanna et al., 2020).
The hippocampus is another area well known for its ability to display experience-driven neuroplastic modifications mediating adaptive behavioral changes (Alberini & Kandel, 2014), its key role in learning and memory (Eichenbaum et al., 1992;Jarrard, 1993), response to stress (Duman et al., 1999;Floriou-Servou et al., 2018) and motivated behavior as well (Bagot et al., 2015;Goto & Grace, 2008;LeGates et al., 2018). In the rat, this brain area, though displaying a highly conserved intrinsic organization, can be divided along its septo-temporal axis into the dorsal (dHC) and ventral hippocampal (vHC) subregions whose functional role, due to their distinct afferent and efferent projections, varies from a preferential involvement of the dHC in the processing of spatial-temporal navigation to a preferential role of the vHC in the processing of emotional memories and related behaviors (Fanselow & Dong, 2010;Tanti et al., 2013). Nonetheless, to our knowledge, only a few studies investigated the effect of sexual activity on hippocampal neuroplasticity, revealing that sexual behavior can induce neurogenesis and is also able to stimulate the growth of dendritic spines, thus inducing a rearrangement of the hippocampal dendritic architecture (Bedos et al., 2018;Glasper & Gould, 2013;Leal-Galicia et al., 2019;. However, the molecular mechanisms at the basis of these effects of sexual activity are still unknown. To obtain further information on whether sexual activity activates neuroplastic processes in the hippocampus, we measured the levels of c-Fos, ΔFosB, BDNF, trkB, and Arc, in the dHC and vHC of control (no sexual behavior), sexually naïve (never exposed before to sexual stimuli-after the first copulatory test) and experienced (i.e., after five copulatory tests) male RHA and RLA rats using Western Blot and/or immunohistochemistry. We then verified whether the levels and changes of these markers were related to the well-known differences in the acquisition of sexual experience, and in sexual motivation and copulatory performance displayed by the two Roman lines .
Since RHA and RLA rats were expected to display the already known differences in copulatory behavior (RHA rats display a more rapid acquisition of sexual experience, e.g., a stable level of sexual activity, and higher levels of sexual motivation and performance, i.e., shorter latencies to mount, intromit and ejaculate, and a higher intromission ratio and ejaculatory frequency than RLA rats), we hypothesized that: (i) these sexual differences should occur concomitantly with line-related differences in the expression of one or more of the above markers in the dHC and/or vHC (e.g., RHA should be expected to display greater experience-induced differences than RLA rats and, in particular, higher levels of c-Fos, ΔFosB, BDNF, and Arc), and (ii) naïve rats of both Roman lines should display greater changes compared to the experienced ones .

| Animals
Outbred male RHA and RLA rats (n = 45 for each rat line) weighing ≈300 g at the beginning of the experiments were from the colony established in 1998 at the University of Cagliari, Italy (Giorgi et al., 2007). Details on the procedures used for the selective breeding of the Sardinian colony are in Giorgi et al. (2005).

| Experimental groups
Sexually naïve male RHA and RLA rats (n = 15 for each line) were rats never exposed before the experiment to a sexually receptive female rat and/or sexual stimuli. Sexually experienced male RHA and RLA rats (n = 15 for each line) were rats that underwent five consecutive 60 min copulatory tests at 3-day intervals (Sanna et al., 2015Sanna, Bratzu, Piludu, et al., 2017; (Contini et al., 2018). In agreement with our previous studies (Sanna et al., 2015Sanna, Bratzu, Piludu, et al., 2017;, after five preliminary tests, rats displayed stable levels of copulatory activity with a receptive female rat (e.g., they become sexually experienced). To determine the basal expression of the molecular markers by Western Blot and immunohistochemistry, age-matched rats of both RHA and RLA lines (n = 15 for each line) never exposed to sexual stimuli and/or copulatory tests were used as controls (see below).

| Sexual behavior
During the copulatory tests, several parameters of sexual motivation and copulatory performance were recorded by an observer who was not aware of the experimental conditions (i.e., who was not aware of the line and the level of sexual experience of the animals). Briefly, the following parameters referred to the first series of copulatory activity

| Tissue sampling
Ninety to 120 min after the end of the copulatory tests (e.g., after the first or the fifth copulatory test for sexually naïve and experienced rats, respectively), male rats were deeply anesthetized with  immunohistochemistry, the perfused brains were fixed by overnight immersion in freshly prepared 4% phosphate-buffered formaldehyde, pH 7.3, at 4 C, as previously described (Quartu et al., 2010;Serra et al., 2017), and then rinsed until they sank in 0.1 M PB, pH 7.3, containing 20% sucrose.

| Western blot
Determination of total protein concentrations was carried out according to Lowry's method (Lowry et al., 1951  Slides were observed with an Olympus BX61 microscope connected to a Leica DFC450C camera for the digital acquisition of images.

| Image densitometry
For the quantitative evaluation of BDNF, trkB, c-Fos, and Arc immunohistochemical labeling, 10Â magnification representative microscopic fields, taken from six animals for each experimental group, scoring 12 different microscopic fields per brain region, were blindly analyzed with ImageJ (http://rsb.info.nih.gov/ij/; RRID:SCR_003070) to calculate the density of immunoreactivity per μm 2 . Mean gray values from unstained areas were subtracted from the gray values of the immunostained regions to exclude background staining.

| Statistical analyses
Statistical analyses of molecular (BDNF, trkB, c-Fos, ΔFosB, and Arc) and behavioral (ML, IL, EL, MF, IF, TMF, TIF, TEF, and PEI) data were conducted in each experimental subject, including those animals which did not display mounts, intromissions or ejaculations. This was done in the attempt to better ascertain whether the differences in sexual behavior between the Roman lines may correlate with differences in the expression of the molecular markers under investigation during the acquisition of sexual experience. Briefly, rats received the full range scores of 900 s if they did not display mounts and/or intromissions with the available female within the first 15 min from the begin of the test; 1800 s if they did not reach ejaculation within 30 min after the first intromission and 600 s if they did not mount/intromit within 10 min after the first ejaculation (Sanna et al., 2015(Sanna et al., , 2020Sanna, Bratzu, Piludu, et al., 2017;. Behavioral data obtained from naïve and sexually experienced male RHA and RLA rats during the first series of copulatory activity (i.e., from the first mount/intromission to the first mount/intromission after the first ejaculation, see Figure 1) or during the whole 60 min experiment (see Figure 2), were analyzed by two-way analyses of variance (ANOVAs) (between-subjects factors: rat line, level of sexual experience; within-subjects factor: test) (see Table 1).
The data from the densitometric analyses of the immunochemical labeling for markers investigated (see Figures 3-7) were also analyzed by two-way ANOVAs (between-subjects factors: rat line, level of sexual experience) (see Table 2 and Table 3). However, in this case, the data obtained from a control group (i.e., a group of rats never exposed to sexual stimuli and/or copulation) were added. 3.2 | Effect of sexual activity on the expression of BDNF, trkB, ΔFosB, and Arc proteins measured by Western blot in the dorsal and ventral hippocampus of sexually naïve and experienced male Roman rats The antibodies against BDNF, trkB, ΔFosB, and Arc recognized respectively a protein band with a relative MW of ffi13 kDa, in agreement with the reported MW of the monomeric form of the BDNF protein (Rosenthal et al., 1991), a band of ffi140 kDa, consistent with the reported MW of the trkB receptor protein (Klein et al., 1989), a band of ffi36 kDa, consistent with the MW of the short isoform of the FosB protein (Tulchinsky, 2000),  Table 2 for F and p values from general ANOVAs). Moreover, pairwise comparisons revealed that BDNF and Arc were increased in naïve RHA rats compared to their controls (+53% and +176%, respectively) and ΔFosB was increased in experienced compared to both control and naïve rats (+56%, in both cases), while no significant differences due to sexual activity were detected in RLA rats. In addition, significant line differences between RHA and RLA rats were also detected, in particular in the naïve condition for Arc expression (+217% RHA vs. RLA) (see Figure 3a for the significance level of single points).
As regards the vHC (Figure 3b), significant main effects of experience were detected for BDNF and ΔFosB expression (see Table 2 for  formation. As a rule, the immunoreactive structures looked denser in the RHA than in the RLA rats and in the dHC than in the vHC.  Table 3 for F and p values from general ANOVAs).
As regards the densitometric analysis, in the dHC line differences between RHA and RLA rats were detected in the whole hippocampal formation and an effect of experience was found in the CA2 and CA3 sectors and in the DG (Figure 4y-b 0 ). Additionally, pairwise comparisons revealed higher BDNF expression levels in naïve RHA rats compared to their controls in the CA3 and the DG (+69% and +63%, respectively); as regards the line differences, a higher BDNF expression was revealed in the CA1, CA2, and CA3 sectors and in the DG of naïve RHA compared to naïve RLA rats (+121%, +168%, +195%, and +151%, in RHA vs. RLA rats, respectively), as well as in the CA1 and CA3 sectors and in the DG of experienced RHA rats compared to their RLA counterparts (+207%, +409%, and +196%, in RHA vs. RLA rats, respectively); in addition, higher BDNF relative levels were found in the CA2 and CA3 sectors and in the DG of control RHA compared to control RLA rats (+403%, +170%, and +128%, in RHA vs. RLA rats, respectively) (see Figure 4y-b 1 for the significance level of single points).
In the vHC, the ANOVAs showed an effect of experience in the CA1 and CA3 sectors and in the DG, and a significant effect of line in the CA1 sector. Additionally, pairwise comparisons revealed in the RHA line a significantly higher BDNF expression in the CA3 sector in experienced compared to naïve rats (+51%) and a lower BDNF expression in naïve than in control rats in the DG (À35%) (see Figure 4s 1 -u 1 for the significance level of single points).

| trkB
The trkB-labeled structures were also represented mostly by nerve fiber systems, having the aspect of filaments, hollow tubules, and coarse punctate elements (Figure 5a-x, a 1 -r 1 ), distributed in between neurons of the pyramidal layer and in the molecular layer of the Ammon's horn, in the alveus, and in between the neuronal cells of the granular layer, in the molecular layer and hilus of the DG (Figure 5a-x, a 1 -r 1 ). In the Ammon's horn, trkB-positive neuronal cell bodies, localized in the pyramidal layer, with labeled proximal processes extending throughout the molecular layer, and in the oriens layer were also observed. In the DG, trkB-immunostained neuronal perikarya were observed in the hilus (Figure 5a-x, a 1 -r 1 ).
As regards the densitometric analysis, in the dHC significant effects of line were detected in all the sectors of the Ammon's horn and a significant effect of experience was found in the DG (see Table 3 for F and p values from general ANOVAs) (Figure 5y-b 0 ). Additionally, pairwise comparisons revealed significant line differences between controls in the CA2 sector (+71% RHA vs. RLA), between naïve animals in the CA3 sector and the DG (+116% and +46%, in RHA vs. RLA rats, respectively), and between the experienced rats in the CA3 sector (+92% RHA vs. RLA).
In the vHC, only an effect of experience was observed in the DG (see Table 3 for F and p values from general ANOVAs) and pairwise comparisons revealed in the RHA line a lower trkB expression in naïve rats compared to controls in the DG (À30%) (see Figure 5s 1 -u 1 for the significance level of single points).

| c-Fos
The c-Fos-immunolabeling was localized to a number of neuronal nuclei in the pyramidal layer of the Ammon's horn and in the granular layer of the DG (Figure 6a-x, a 1 -r 1 ).
As regards the densitometric analysis, in the dHC significant effect of experience was detected in all considered subregions while significant effect of line was detected in the CA2 and CA3 sectors and significant line Â level of sexual experience interactions in the CA1 and CA3 sectors of the Ammon's horn. Moreover, pairwise comparisons revealed that, in the Ammon's horn, the CA1 sector displayed significant higher expression of c-Fos in naïve RHA rats compared to both controls and experienced ones (+150% and +899%, respectively), while the CA2 and CA3 sectors of the RHA line, and the CA1 sector of the RLA line showed significant higher expression in naïve than in experienced rats (+285%, +1286%, and +326%, respectively). At variance, in the DG, both Roman rat lines displayed significant higher expression in naïve rats compared to controls and experienced ones (by about +165% and +263%, for RHA and RLA rats, respectively). Finally, significant line-dependent differences were observed between naïve RHA and RLA rats in all sectors of the Ammon's horn with RHA rats displaying a higher c-Fos expression F I G U R E 4 Regional distribution (a-x, a 1 -r 1 ) and densitometric analysis (y-b 0 , s 1 -u 1 ) of changes induced by sexual activity in the BDNF-like immunoreactivity (LI) in coronal sections of the dorsal and ventral hippocampus of male RHA and RLA rats under control (Ctrl) (no copulation), sexually naïve (Naïve) (after the first copulatory test), and experienced (Exp) (after five copulatory tests) conditions. (a-f, a 1 -f 1 ) CA1 sector; (g-l) CA2 sector; (m-r, g 1 -l 1 ) CA3 sector of the Ammon's horn; (s-x, m 1 -r 1 ) dentate gyrus (DG). Dashed lines mark the boundaries of pyramidal (p) and granular (g) layers. (y-b 0 , s 1 -u 1 ) Densitometric analysis; values are mean ± SEM of six rats for each experimental group obtained by averaging the optical density (OD) from 12 different microscopic fields for each brain region. Two-way ANOVAs followed by pairwise comparisons. E, significant effect of level of sexual experience; L, significant effect of line. *p < .05; **p < .01; ***p < .001. h, hilus; m, molecular layer. Scale bars: a-x = a 1 -r 1 = 50 μm than their RLA counterparts (with differences from +79% to +180%) (see Figure 6y-b 0 for the significance level of single points).
In the vHC, significant effects of line and experience as well as significant line Â experience interactions were detected in the CA1 and CA3 sectors and in the DG (see Table 3 for F and p values from general ANOVAs) and, similarly to what seen in the dHC, additional pairwise comparisons showed that naïve RHA rats displayed higher expression than both control and experienced animals in the Ammon's horn and DG (with differences from +96% to +803%) as well as a higher c-Fos expression compared to their RLA counterparts (with differences from +157% to +341%). In contrast, no significant experience-related differences were observed in RLA rats (see Figure 6s 1 -u 1 for the significance level of single points).

| Arc
The Arc immunostaining occurred in neuronal perikarya in the Ammon's horn and the DG (Figure 7a-x, a 1 -r 1 ).
As regards the densitometric analysis, effects of experience were detected in the CA1 and CA3 sectors and in the DG of the dHC, where an effect of line and a significant interaction were also detected (see Table 3 for F and p values from general ANOVAs).
Moreover, pairwise comparisons revealed higher Arc expression in naïve than in controls and experienced RHA rats in the CA3 and DG (with increases of by about +64% and +300% in the CA3 and DG, respectively) and higher Arc expression in naïve RHA compared to their RLA counterparts in the DG (+225%) (see Figure 7y-b 0 for the significance level of single points).
In the vHC, the effect of experience for Arc expression was significant in both the Ammon's horn and the DG (see Table 3 for F and p values from general ANOVAs); additionally, pairwise comparisons detected lower Arc expression in naïve RHA rats compared to control (À52%) and experienced rats (À53%) in the CA1 sector and to controls in the DG (À52%); similarly, in the RLA line naïve rats displayed lower Arc expression in the CA1 and CA3 sectors when compared to the experienced ones (À49% and À46%, respectively) (see Figure 7s 1 -u 1 for the significance level of single points).

| DISCUSSION
To our knowledge, this study shows for the first time that sexual activity (60 min of copulatory activity with a receptive female rat) induces differential changes in the expression levels of putative molecular markers of neural activation, such as c-Fos and ΔFosB, and of neural plasticity, such as BDNF, trkB, and Arc in the dorsal and ventral subdivisions of the hippocampus of sexually naïve and experienced RHA and RLA rats, when measured either in tissue homogenates using Western Blot or within their sub-regions, that is, DG, CA1, CA2, and CA3, through densitometric analysis of immunolabeled tissue sections. When present, the changes (increases or decreases) were usually more evident in RHA than in RLA rats, and in particular when rats of both lines were in the naïve rather than in the experienced condition, and in the dorsal rather than in the ventral subdivision of the hippocampus. The differential changes in the expression of BDNF, trkB, c-Fos, ΔFosB, and Arc in the dHC and vHC of sexually naïve and experienced RHA and RLA rats occured in parallel with the well-known differences observed in sexual behavior between the two rat lines-RHA rats exhibit higher sexual motivation and better copulatory performances than RLA rats-which are secondary to the higher mesocorticolimbic dopaminergic tone present in RHA versus RLA rats (Sanna et al., 2015Sanna, Bratzu, Piludu, et al., 2017;.

| BDNF/trkB
As extensively shown in Section 3, sexual activity influences BDNF levels in the dHC and vHC and their sub-regions depending on the rat line, the level of sexual experience, and the hippocampal subdivision considered. The results suggest that sexual activity has opposite effects on BDNF levels in the dHC and vHC and their subregions.
Indeed, the BDNF levels measured by Western blot increased in the dHC of both RHA and RLA naïve rats, while they decreased in the vHC of sexually naïve rats of both rat lines compared to control rats.
The majority of the above changes are confirmed by immunohistochemistry followed by densitometric analysis. The reasons for these opposite changes are unknown. However, it is reasonable to assume that they reflect the specific functional roles of the two partitions of the hippocampus, the dHC playing the main role in cognitive functions such as learning and memory, and the vHC in anxiety, stress, and affection (Fanselow & Dong, 2010;Tanti et al., 2013;and references therein).
The densitometric analysis also revealed that the opposite changes induced by sexual activity in the dHC and vHC on BDNF expression occur with several differences among the hippocampal subregions in the RHA versus the RLA rats, either in controls or in F I G U R E 5 Regional distribution (a-x, a 1 -r 1 ) and densitometric analysis (y-b 0 , s 1 -u 1 ) of changes induced by sexual activity in the trkB-like immunoreactivity (LI) in coronal sections of the dorsal and ventral hippocampus of male RHA and RLA rats under control (Ctrl) (no copulation), sexually naïve (Naïve) (after the first copulatory test), and experienced (Exp) (after five copulatory tests) conditions. (a-f, a 1 -f 1 ) CA1 sector; (g-l) CA2 sector; (m-r, g 1 -l 1 ) CA3 sector of the Ammon's horn; (s-x, m 1 -r 1 ) dentate gyrus (DG). Dashed lines mark the boundaries of pyramidal (p) and granular (g) layers. (y-b 0 , s 1 -u 1 ) Densitometric analysis; values are mean ± SEM of six rats for each experimental group obtained by averaging the optical density (OD) from 12 different microscopic fields for each brain region. Two-way ANOVA followed by pairwise comparisons. E, significant effect of level of sexual experience; L, significant effect of line. *p < .05; **p < .01; ***p < .001. h, hilus; m, molecular layer. Scale bars: a-x = a 1r 1 = 50 μm sexually naïve and experienced rats. In this regard, it is noteworthy that in the dHC, sexual interaction leads in the RHA, but not RLA, rats to an increase of the BDNF expression that occurs mainly in the CA3 sector and the DG; by contrast, a different trend occurs in the vHC, where BDNF is expressed at lower levels in the DG of sexually naïve RHA rats compared to controls and shows higher levels in the CA3 sector of sexually experienced RHA rats compared to their naïve counterparts. The CA3 sector receives the mossy fiber projections, and undergoes a continuous dynamic adjustment of its connectivity, together with processes such as synaptogenesis and dynamic structural plasticity of its pyramidal dendritic arborizations Seki & Rutishauser, 1998;Uysal et al., 2012). Thus, it is tempting to speculate that the differential changes in BDNF levels (that occur together with changes in the levels of its trkB receptor, see below) that occur in the dHC and vHC may be related to the differential changes in the adjustments of synaptic connectivity occurring in the dHC and VHC of the two Roman rat lines, which should be expected to be maximal at the first sexual interaction with a receptive female rat and to undergo adaptive changes with the repetition of sexual activity, which leads to the acquisition of sexual experience.
This is very evident in RHA rats, but not in RLA rats in which changes in BDNF expression induced by sexual activity are very modest when compared to those observed in RHA rats.
Unfortunately, these findings do not help in clarifying whether the generally lower levels of BDNF-like immunoreactivity found in RLA rats in this and other studies Serra et al., 2017Serra et al., , 2018 are due to a slower synthesis rate of the BDNF protein or to other causes in this Roman rat line compared to the RHA counterpart. In fact, it is also plausible that the presumably slower synthesis of the BDNF protein in the RLA rats may, in turn, lead to a deficit in the synaptic release and reduced target-derived support to promote the synaptic contacts with the mossy fibers (Isgor et al., 2015). Nonetheless, this finding suggests that an impaired activity of the BDNF/trkB system might be involved in the different behavioral responses of RHA and RLA when exposed to a sexually receptive female rat  or to a 15 min forced swimming test (Serra et al., 2018). The differential changes induced by sexual activity in BDNF levels in the dHC and vHC of RHA versus RLA rats occur together with differences in the levels of trkB, the highaffinity BDNF receptor (Klein et al., 1989), especially in the dHC, showing in RLA rats trkB levels lower than those of RHA rats. In this regard, it is known that the silencing of trkB receptors in the mPOA decreased consummatory sexual behavior (Brague et al., 2018) and that administration of a trkB antagonist induces lower initial levels of sexual motivation (Hawley & Mosura, 2019).
However, the relation between the differential trkB and BDNF levels in the dHC (and to a lesser extent in the vHC) and the differences in sexual behavior between the two Roman rat lines remain issues to be further investigated, for instance by carrying out experiments involving a direct manipulation of the activity of the trkB receptor and/or of the BDNF-mediated transmission.

| C-Fos
This study shows that sexual activity also influences c-Fos levels measured by immunohistochemistry followed by densitometry in the dHC and vHC and their subregions depending on the rat line, the level of sexual experience, and the hippocampal subdivision. Accordingly, c-Fos levels were increased after sexual activity in the dHC and vHC of both sexually naïve, but not experienced RHA and RLA rats, when compared to control rats, and the higher increases were found in the DG and CA1 subregions followed by the CA2 and CA3 ones of sexually naïve RHA versus RLA rats. However, in spite of the fact that c-Fos increased in the dHC and vHC of both sexually naïve RHA and RLA rats and that in the DG of the dHC of both RHA and RLA rats c-Fos levels were very similar, c-Fos increases were usually higher in both the dHC and vHC in RHA than RLA rats, in which c-Fos increased only in the dHC. The reason for these differences between sexually naïve RHA and RLA is unknown. Regardless these differences, the similar activation in the DG of dHC in both Roman lines lead to speculate that the first exposition to a sexually receptive female targets discrete cell groups that, independently from the rat line, may represent the hippocampal contribution to reward memory (Gauthier & Tank, 2018;Gava et al., 2021;Schuette et al., 2020).
Accordingly, our findings in naïve animals remind the trend of c-Fos activation upon exposure to environmental novelty (Snyder et al., 2009).
Irrespective of the significance of the differential changes in c-Fos levels between the Roman lines, the c-Fos increase after sexual activity in the hippocampus of sexually naïve but not experienced rats resembles the trend of c-Fos increases that occurs in the VTA, the Acb, and the mPFC of sexually naïve but not experienced RHA and RLA rats . These findings suggest that once the sexual experience has been acquired, the raise of c-Fos levels is not further required for, nor is involved in, the activation of mechanisms F I G U R E 6 Regional distribution (a-x, a 1 -r 1 ) and densitometric analysis (y-b 0 , s 1 -u 1 ) of changes induced by sexual activity in the c-Fos-like immunoreactivity (LI) in coronal sections of the dorsal and ventral hippocampus of male RHA and RLA rats under control (Ctrl) (no copulation), sexually naïve (Naïve) (after the first copulatory test), and experienced (Exp) (after five copulatory tests) conditions. (a-f, a 1 -f 1 ) CA1 sector; (g-l) CA2 sector; (m-r, g 1 -l 1 ) CA3 sector of the Ammon's horn; (s-x, m 1 -r 1 ) dentate gyrus (DG). Dashed lines mark the boundaries of pyramidal (p) and granular (g) layers. (y-b 0 , s 1 -u 1 ) Densitometric analysis; values are mean ± SEM of six rats for each experimental group obtained by averaging the optical density (OD) from 12 different microscopic fields for each brain region. Two-way ANOVA followed by pairwise comparisons. E, significant effect of level of sexual experience; L, significant effect of line. *p < .05; **p < .01; ***p < .001. h, hilus; m, molecular layer. Scale bars: a-x = a 1r 1 = 50 μm F I G U R E 7 Legend on next page. SANNA ET AL.
facilitating sexual performance and might be secondary to a possible repressive effect of accumulated ΔFosB protein on c-Fos expression (Renthal et al., 2008;Ruffle, 2014) (see also ΔFosB paragraph below) in experienced animals, as already extensively discussed . Although further studies are required to clarify this point, these results show that the hippocampus has to be added to the list of brain areas where c-Fos levels are increased upon the sexual activity, such as the MPOA, the PVN, the bed nucleus of the stria terminalis (BNST), the medial amygdala, the piriform cortex, the VTA, the Acb, and the mPFC (Beloate et al., 2016;Biały & Kaczmarek, 1996;Bradley & Meisel, 2001;Nishitani et al., 2004;Nutsch et al., 2016;Pitchers, Frohmader, et al., 2010;Veening & Coolen, 1998;Witt & Insel, 1994).

| ΔFosB
Sexual activity also influences differentially the levels of ΔFosB, a stable truncated form of the FosB protein that tends to accumulate in brain tissues (Nestler, 2008), in the dHC and vHC of the Roman lines.
In fact, ΔFosB increased mainly in both the dHC and vHC of sexually experienced but not naïve RHA rats, with a similar not significant increase tendency in the vHC but not in the dHC of sexually experienced RLA rats. The ΔFosB increase found in the hippocampus of sexually experienced, but not naïve, RHA rats confirms that repeated sexual activity is necessary to increase ΔFosB levels in areas of interest for sexual behavior such as the Acb, VTA, mPFC, mPOA, and caudate-putamen (Hedges et al., 2009;McHenry et al., 2016;Pitchers, Frohmader, et al., 2010;Sanna et al., 2019;Wallace et al., 2008). These findings are in line with the hypothesis that ΔFosB is a reliable marker of neural activation linked to the acquisition of sexually rewarding experience (Pitchers et al., 2013) and that its expression in the limbic system drives the reward and sexual experience-induced facilitation of sexual performance (Hedges et al., 2009;Pitchers, Frohmader, et al., 2010;Wallace et al., 2008).
However, as found in the VTA, Acb, and mPFC of sexually experienced RLA versus RHA rats , the above conclusions are complicated by the very modest ΔFosB increase that occurs in the hippocampus of sexually experienced RLA rats.
The reason why repeated sexual activity increases ΔFosB levels in the hippocampus of RHA much more than in RLA rats is unknown.
One explanation may be that the dopaminergic tone generated by VTA neurons that innervate the hippocampus (Gasbarri et al., 1997), which is also very rich in dopaminergic receptors (see Eagle et al., 2015;Kempadoo et al., 2016), is stronger in RHA than RLA rats, as already proposed and extensively discussed for other limbic areas (Acb and mPFC) of the two rat lines. In line with this explanation (i) dopamine from the VTA may regulate the induction of ΔFosB providing a message to hippocampal neurons that relate to the salience and novelty of events (Lisman & Grace, 2005), (ii) dopamine enhances plasticity in CA1 pyramidal neurons (Li et al., 2003) and mediates network-level activity and memory persistence (McNamara et al., 2014), and (iii) the higher mesocorticolimbic dopaminergic tone of RHA versus RLA rats has been already considered responsible of F I G U R E 7 Regional distribution (a-x, a 1 -r 1 ) and densitometric analysis (y-b 0 , s 1 -u 1 ) of changes induced by sexual activity in the Arc-like immunoreactivity (LI) in coronal sections of the dorsal and ventral hippocampus of male RHA and RLA rats under control (Ctrl) (no copulation), sexually naïve (Naïve) (after the first copulatory test), and experienced (Exp) (after five copulatory tests) conditions. (a-f, a 1 -f 1 ) CA1 sector; (g-l) CA2 sector; (m-r, g 1 -l 1 ) CA3 sector of the Ammon's horn; (s-x, m 1 -r 1 ) dentate gyrus (DG). Dashed lines mark the boundaries of pyramidal (p) and granular (g) layers. (y-b 0 , s 1 -u 1 ) Densitometric analysis; values are mean ± SEM of six rats for each experimental group obtained by averaging the optical density (OD) from 12 different microscopic fields for each brain region. Two-way ANOVA followed by pairwise comparisons. E, significant effect of level of sexual experience; L, significant effect of line. *p < .05; **p < .01; ***p < .001. h, hilus; m, molecular layer. Scale bars: a-x = a 1r 1 = 50 μm Note: F values and significance levels from two-way ANOVAs (df = 2, 1, 2, 42) performed on the relative BDNF, trkB, ΔFosB, and Arc protein levels found in control (no copulation), sexually naïve (after one copulatory test) and sexually experienced (after five copulatory tests) male rats by means of Western blot analysis from the experiments shown in Figure 3. Abbreviations: dHC, dorsal hippocampus; ns, not significant; vHC, ventral hippocampus.
the higher increase of ΔFosB levels found in the VTA, Acb, and mPFC of sexually experienced RHA versus RLA rats . The assumption that the higher levels of ΔFosB in the dHC of RHA versus RLA rats are secondary to a higher dopaminergic tone deriving from the VTA-hippocampal projection also provides an indication of the still unknown sexual role(s) of ΔFosB expression in the hippocampus.
Accordingly, in line with the higher dopaminergic tone of RHA rats and with the recognized role of the dHC in cognitive functions, in particular learning and memory (Fanselow & Dong, 2010), it is reasonable that the higher levels of ΔFosB in the dHC of sexually experienced RHA rats play a role in the faster acquisition of sexual experience by, and/or stabilization of sexual performance in RHA vs. RLA rats. Conversely, since the vHC plays a main role in anxiety, response to stress and emotion (Fanselow & Dong, 2010), and this occurs together with its interaction with the Acb and the mPFC (see Eagle et al., 2020), the differences in ΔFosB levels in this area might play a more general role in the differences in behavioral traits of the two Roman rat lines, including those in sexual behavior (see above), rather than a specific role in the process of acquisition/stabilization of sexual experience.
Further studies are required for testing these hypotheses.

| Arc
Sexual activity also influences the levels of the Arc protein in the dHC and vHC and their subregions. Accordingly, Arc levels measured by Western Blot increased in the dHC of sexually naïve RHA and RLA rats compared to controls and sexually experienced counterparts. The T A B L E 3 Effect of sexual experience on Roman high and low avoidance rat lines  (Bramham et al., 2010;Korb & Finkbeiner, 2011;Moga et al., 2004;Shepherd & Bear, 2011), as it may be considered the first exposition to, and copulation with a sexually receptive female rat. Thus, once sexual experience has been acquired and Arc has produced the plastic changes necessary to maintain it, it appears reasonable that Arc content return to its basal (control) values. More difficult is to explain why Arc decreases in the vHC of sexually naïve RHA and RLA rats when compared to controls, a decrease that disappears in sexually experienced rats. Although these findings might be related to the differences in the functional roles of the two hippocampal sub-divisions (the dHC playing a main role in learning and memory and the vHC in anxiety, stress, and affection, see Fanselow & Dong, 2010), the specific reasons for the observed differential Arc expression in the dHC and vHC after the first sexual intercourse are still unknown. However, since a similar difference occurs also in BDNF expression (BDNF increases in the dHC and decreases in the vHC of sexually naïve RHA and RLA rats, see above), given that BDNF has been found able to regulate Arc activity and expression in neuronal dendrites to produce cytoskeleton rearrangements indispensable for BDNF induced long-term potentiation in the hippocampus (Cohen et al., 2011;Leal et al., 2015;Messaoudi et al., 2007), it is tempting to speculate that the higher and lower BDNF levels may be involved in the higher and lower Arc levels found in the dHC and vHC of sexually naive RHA and RLA rats, respectively, and that these changes disappear in sexually experienced RHA and RLA rats after the acquisition of sexual experience. Further studies are required to verify this hypothesis.

| Final remarks
This study shows that 60 min of copulatory activity induces a series of differential changes in the expression c-Fos and ΔFosB, Arc, and BDNF/trkB in the dHC and vHC of male RHA and RLA rats, which show different patterns of copulatory behavior . These differences depend on (i) the rat line (RHA vs. RLA); (ii) the level of sexual experience (controls, naïve and experienced); (iii) the hippocampal subdivision and their relevant sub-regions (dorsal vs. ventral DG, CA1, CA2, and CA3); and (iv) the marker considered. Indeed, when present, the differences were usually more evident in RHA than RLA rats, in the naïve rather than in the experienced condition, and in the dHC rather than in the vHC. The differential changes in BDNF, trkB, c-Fos, ΔFosB, and Arc levels in the dHC and vHC of sexually naïve and experienced RHA and RLA rats occurred in parallel with the differential changes in sexual behavior of the two rat lines, which are more evident in the naïve condition and tend to decrease but do not disappear in the experienced condition . These findings confirm that sexual activity induces a neural activation, possibly mediated by c-Fos and ΔFosB, that leads to trophic and plastic changes mediated by BDNF/trkB and Arc at the level of the dHC and vHC. These changes become stable with repetition of sexual activity, which probably lasts until sexual activity persists. In line with this hypothesis sexual behavior has been found able to induce neurogenesis in the hippocampus of young and middle-aged male adult rats (Bedos et al., 2018;Glasper & Gould, 2013;Leal-Galicia et al., 2019; and when repeated along time, to stimulate the growth of dendritic spines, leading to a rearrangement of the dendritic architecture in the hippocampus , with structural changes that persist until sexual activity is continued in the experimental period (Glasper & Gould, 2013). The results of this study suggest that the stable morphological modifications induced by sexual activity found in the above studies may be secondary to changes in neuronal activation in which c-Fos and ΔFosB play a key role, and which lead to changes in the trophic and plastic activities of the BDNF/trkB and Arc systems, which occur in the dHC and vHC mainly during the first sexual experience(s). More importantly, the above changes depend on the animal genotypic/phenotypic characteristics, being more evident in RHA than in RLA rats, a finding that is parallel to the higher sexual motivation and better sexual performance of RHA versus RLA rats.
Further studies are required to verify whether the sexual activityinduced differential changes in BDNF, trkB, and Arc levels in the dHC and vHC are causal and/or consequent to the higher sexual motivation and performance of RHA versus RLA rats. Moreover, it is likely that the changes in the expression of BDNF, trkB, c-Fos, ΔFosB, and Arc between the sexually naïve and the experienced Roman rats that occur in the hippocampus, and in the limbic system in general, may all play a role in the characteristic Roman line differences not only in sexual behavior but also in other motivated behaviors, and their alterations (Fernández-Teruel et al., 2021;Giorgi et al., 2007Giorgi et al., , 2019Melis et al., 2019;Serra et al., 2018).
Finally, this study also confirms that the hippocampus participates in a complex limbic brain circuit connecting areas such as the VTA, Acb, amygdala, and BNST to hypothalamic nuclei (PVN, mPOA) in which neurotransmitters as dopamine and excitatory amino acids, and neuropeptides as oxytocin contribute to mediate the interaction between motivational and consummatory aspects of male sexual behavior Melis et al., 2007Melis et al., , 2009Melis et al., , 2010, 2021a, 2021bSanna et al., 2020;Succu et al., 2008Succu et al., , 2011. Noteworthy, after sexual activity, differential changes in the expression of BDNF, trkB, c-Fos, ΔFosB, and Arc, which resemble those found in the hippocampus in this study, also occur in the VTA, Acb and mPFC of sexually naïve and experienced RHA and RLA rats .
In conclusion, sexual activity induces differential changes in the expression of BDNF, trkB, c-Fos, ΔFosB, and Arc proteins in the dHC and vHC of RHA and RLA rats. While the changes in the dHC and vHC are likely to reflect the different specialization of the two hippocampal subdivisions, the more robust protein level changes in RHA versus RLA rats, which occur in particular after the first sexual experience, appear mainly related to the intrinsically different genotype/ phenotype of the two Roman rat lines, which leads to differences in the plastic processes at the basis of the acquisition of sexual experience taking place in the hippocampus and other limbic brain areas.


s2fieldsofstudy: Biology Medicine

text:
HIV Pre-exposure Prophylaxis and Its Impact on the Gut Microbiome in Men Having Sex With Men

HIV/AIDS still imposes a high epidemiological and societal burden. Together with antiretroviral therapy, pre-exposure prophylaxis (PrEP) represents a fundamental tool in the fight against HIV/AIDS. PrEP is considered effective and safe, even though it may affect organs like the kidney, bone, and liver, as shown by randomized clinical trials (RCTs). These side effects may be mediated by alterations of the gut microbiome. Whilst the impact of the human rectal and vaginal microbiome on HIV prevention has been highly investigated among women, less is known about its effect among men having sex with men (MSM), a vulnerable population at high risk for HIV and disproportionately affected by HIV/AIDS. In the present paper, we will overview the effects of PrEP on the gut microbiota in MSM. Mining PubMed/MEDLINE, we identified three studies that have found significant changes affecting the gut microbiota. However, these shifts in the gut microbiome composition are variable, probably due to methodological differences, even though all studies reviewed in the present overview consistently report aberrations at the level of the gut microbiota. More data are needed, especially concerning the long-term side effects of PrEP: despite the studies included being a high-quality RCT, and two well-designed cross-sectional studies, evidence related to the impact of HIV PrEP on the gut microbiome in MSM is scarce and based on small populations. A better understanding of the interactions between the gut microbiota, sexual orientation/identity, and HIV prevention is expected to improve PrEP adherence and devise strategies to counteract PrEP-related side effects.

HIV prevalence is particularly high in low-resource settings and in southern sub-Saharan Africa, with a highly variable coverage of antiretroviral therapy (ART).
Recently, a new technology has been introduced in the pharmacological armamentarium against HIV/AIDS: the so-called pre-exposure prophylaxis, or PrEP.

HIV PRE-EXPOSURE PROPHYLAXIS AND ITS SIDE EFFECTS
Truvada ® , a fixed-dose combination antiretroviral medication for HIV prevention (PrEP), was approved by the United States (US) Food and Drug Administration (FDA) in 2012. Its ingredients are tenofovir disoproxil fumarate (TDF)-a nucleotide analog reverse transcriptase (RT) inhibitor (NARTI/NRTI)and emtricitabine (FTC)-a nucleoside analog reverse RT inhibitor (NtARTI/NtRTI). Descovy ® , approved by the FDA in 2016, is a combination of emtricitabine and tenofovir alafenamide (Spinner et al., 2016).
PrEP, if taken once daily as prescribed, is highly effective in preventing HIV, curbing its risk by more than 90% in men having sex with men (MSM). Together with ART, treatmentas-prevention (TASP), and treat-and-test strategies, PrEP is a fundamental tool in the fight against HIV/AIDS. The roll-out of PrEP has, indeed, contributed to halving new HIV diagnoses in countries like Australia and Scotland (Grulich et al., 2018;Estcourt et al., 2021).
While Truvada ® and Descovy ® are taken orally, Apretude ® represents the third formulation of PrEP, which consists of the long-acting, injectable combination of cabotegravir (an integrase inhibitor) and rilpivirine (a non-nucleoside RT inhibitor, NNRTI), administered every 2 months by a specialized healthcare worker (Kim, 2021). It was recently approved by the FDA, in 2021.
However, despite its high effectiveness, PrEP may have some major side effects, affecting organs like the bone, kidney, and liver. Some randomized clinical trials (RCTs) have shown signs of kidney impairment (with the insurgence of renal tubular toxicity, tubular dysfunction, and phosphaturia; Schaefer et al., 2022), decreased bone mineral density (Spinelli et al., 2019;Baranek et al., 2020), and increased values of hepatic transaminases (Mandala et al., 2014). According to a recently published global PrEP user meta-analysis (Schaefer et al., 2022), pooling together 17 articles and synthesizing 11 RCTs, totaling 13,523 participants, PrEP use was associated with an increased risk of grade 1 and higher kidney adverse events (with an odds-ratio, OR, of 1.49 [95% confidence interval, CI 1.22-1.81]) and grade 2 and higher events (with an OR of 1.75 ). Changes in bone mineral density are variable in the existing scholarly literature, with a substudy of the international, open-label PrEP demonstration project iPrEx-"open-label extension" (OLE) study estimating a 1.2 and 0.5% decrease in the spine and hip bone mineral density, respectively (Spinelli et al., 2019). A recently published systematic review and meta-analysis (Baranek et al., 2020), pooling together 25 studies, has computed a significant bone mineral density drop at the level of the lumbar spine (with a mean difference, MD, of −0.82% [95% from −1.28% to −0.37%]), and hip (with an MD of −0.81% [95%CI from −1.22% to −0.40%]). On the other hand, the use of PrEP was not associated with an increased risk of fracture. Concerning hepatic toxicity (Mandala et al., 2014), this can be mild to moderate, especially in individuals who are hepatitis B virus (HBV) surface antibody (HBsAb) positive or have underlying liver disease.
The cellular and molecular mechanisms of these adverse events are still poorly understood, and given the increasingly prominent role of PrEP in the fight against HIV/AIDS, it is of paramount importance to better understand them. Some scholars hypothesize that reduced bone mineral density may be due to changes in osteoblast gene expression or could occur via osteoprotegerin (OPG)/receptor activator of nuclear factor κΒ (NFκΒ; RANK) ligand (RANKL)/RANK system dysregulation (Brown et al., 2011;Delpino and Quarleri, 2020), whereas tubular injury may be mediated by the expression of the organic anion transporter 1 (OAT1) and the multidrug resistance protein 4 (MRP4) transporter (Kohler et al., 2011).
PrEP may result also in gastrointestinal side effects (especially within the first 3 months of treatment, known as "PrEP start-up syndrome"; Mugwanya and Baeten, 2016), and may alter the gut microbiome, which is functionally linked to the kidney, bone, and liver. Whilst the impact of the human (vaginal and rectal) microbiome on HIV prevention has been highly investigated and reviewed among women (Velloza and Heffron, 2017;Farcasanu and Kwon, 2018;Abdool Karim et al., 2019), less is known about its effect among MSM, despite the fact that this population is particularly at high risk for HIV and is disproportionately affected by HIV/AIDS. To the best of our knowledge, there are no comprehensive reviews covering this important topic. In the present paper, we will briefly overview the effects of the use of PrEP on the gut microbiota in MSM. For this purpose, we have mined PubMed/MEDLINE, the major biomedical database, searching for words such as "gut flora, " "gut microbial communities, " "gut microbiota, " "gut microbiome, " "intestine, " "rectal, " "anal, " "HIV, " "AIDS, " "men having sex with men, " "sexual identity, " "sexual orientation, " "tenofovir, " "emtricitabine, " and "pre-exposure prophylaxis. "

THE GUT MICROBIOME AND ITS IMPACT ON HEALTH AND DISEASE
The gut microbiome is extremely heterogeneous and plastic and consists of a highly diverse population of bacteria that can have both beneficial and detrimental impacts on human health (Fan and Pedersen, 2021). It is composed of more than 1,200 species of bacteria (Rinninella et al., 2019), including Bacteroides, Actinomycetes, Firmicutes, Proteobacteria, and Verrucomicrobia. It plays different functions, ranging from nutrient absorption (in particular, micro-nutrients), and processing to metabolic homeostasis (including favoring insulin sensitivity), and fine-tuning of the immune system, protecting especially newborns from respiratory and intestinal infections and pathogen invasion (Flint et al., 2012). It can also provide the individual with sources of energy, by fermenting and processing short-chain fatty acids (SCFAs), like butyrate, acetate, and propionate (Valdes et al., 2018).
Gut microbiome-axes have also been demonstrated for the numerous organs impacted by PreP. The gut microbiota is dynamically interconnected with the kidney. In individuals with chronic kidney disease (CKD), it may be impaired and produce several uremic toxins and solutes, like p-cresyl sulfate (PCS), trimethylamine (TMA) N-oxide (TMAO), and indoxyl sulfate. In turn, increased urea levels result in gut microbiome alterations. The release of uremic toxic metabolites may lead to renal anemia, asthenia, pruritus, impaired mineral bone density, cardiovascular, and neurological disorders, which are commonly observed in CKD patients as well as in subjects with acute kidney injury (AKI), IgA nephropathy, nephrolithiasis, hypertension, or those needing hemodialysis/peritoneal dialysis (Chen et al., 2019;Hobby et al., 2019;Stavropoulou et al., 2021). Gut microbiota overgrowth or change in profile composition may result in alterations in calcium, vitamin K, and vitamin B levels, as well as in vitamin D absorption and metabolism, leading to fractures and osteoporosis (Ding et al., 2020). Cellular and molecular mechanisms include modulation of insulin-like growth factor (IGF)-1-related cascades. IGF-1 is known to fine-tune the bone cells' differentiation into osteoblasts, osteoclasts, and chondrocytes (Pacifici, 2018). The gut microbiota can also affect the concentrations of vitamin D metabolites, steroid hormones, and the parathyroid hormone (PTH; Pacifici, 2018). A new, high interdisciplinary field is emerging, termed "osteomicrobiology, " to underline the strong links and connections between bone and microbes (Bhardwaj et al., 2022). The gut microbiome also impacts liver functioning. Gut microbiota overgrowth and, in particular, small intestinal bacterial overgrowth (SIBO) of Gram-negative pathogens contribute to reducing overall microbial diversity and to tissue damage, by the production and release of bacterial 16sDNA, ethanol, toxic metabolites, and endotoxins (like TMAO and LPS), which inhibit cholesterol conversion into bile acids, alter intestinal permeability, promote a pro-inflammatory milieu, modulate apoptotic cascades and exacerbate the underlying liver disease (Zhou et al., 2021). Impaired fermentation and processing of SCFAs result in decreased stimulation of gut endocrine cells and reduced secretion of glucagon-like peptide 1 (GLP-1) with a subsequent drop in hepatic fatty acid β-oxidation and lipogenesis. Also, PPARα-mediated β-oxidation of fatty acids is altered, leading to a worsening of hepatosteatosis and liver disease. Moreover, aberrant gut microbiota leads to the activation of hepatic stellate cells (HSCs) via the intrahepatic T-cell receptor (TCR) immune repertoire (IR; TCR-IR; Liang et al., 2020). This results in the shift from a quiescent to an active, proliferative, migratory, and fibrogenic phenotype, transforming the cell into a myofibroblast, contributing to liver fibrogenesis.

THE GUT MICROBIOME AND HIV/AIDS
Among the different diseases, changes in the human microbiome composition have been associated with HIV serostatus (Noguera-Julian et al., 2016;Tuddenham et al., 2020;Zhou et al., 2020), with a few exceptions . Shifts in microbial composition have been linked as well with sexual orientation/ sexual identity (Noguera-Julian et al., 2016;Li et al., 2019;Tuddenham et al., 2020;Zhou et al., 2020), as shown also by animal models . Li et al. (2019) transplanted feces from HIV-negative men having sex with women (MSW) and MSM, as well as from HIV-positive untreated MSM to gnotobiotic mice. After the transplant, the microbiomes of MSM and MSW conserved distinct compositions in mice. From an immunological standpoint, HIV-negative MSM donors exhibited higher frequencies of blood CD38+ HLA-DR+ and CD103+ T-cells whereas their fecal recipients had higher frequencies of gut CD69+ and CD103+ T-cells. Probably due to the small sample size employed, the authors failed to detect statistically significant differences induced by HIV serostatus both at the level of the microbiome and immunologically. On the other hand, after infecting primary human lamina propria cells treated with fecal microbiota, gut flora from MSM caused higher levels of HIV replication. Among humans, HIV-seropositive individuals and MSM consistently report alterations in the gut flora, affecting, for instance, Bacteroides caccae, Bacteroides ovatus, Bacteroides uniformis, and Prevotella stercorea, with markedly reduced richness and alpha diversity of the gut microbiota. This can be accompanied by profound metabolic changes involving several functional pathways (such as those regulating carbohydrate, lipid, and amino acid metabolism), according to a recently published systematic review and meta-analysis of 12 studies (Zhou et al., 2020).
Further, the gut microbiome may increase the risk of contracting HIV (Coleman et al., 2020) via increased expression of integrin and chemokine receptors on T-cells, especially at the level of the colon. Furthermore, it may influence HIV transmission (Coleman et al., 2020), favoring HIV persistence (Koay et al., 2018) through immune activation and systemic inflammation, and impacting the progression from acute HIV Frontiers in Microbiology | www.frontiersin.org infection (AHI; Sortino et al., 2019) to AIDS (Sortino et al., 2019), with microbiota diversity correlating with CD4+ T-cell diversity (Koay et al., 2018;Sortino et al., 2019). As the disease progresses, an increase in pathogenic species, including Proteobacteria, and a decrease in protective ones (like Bacteroides, Lactobacillus, and Bifidobacterium) are observed (Ling et al., 2016;Ribeiro et al., 2017;Vujkovic-Cvijin et al., 2020). The intestinal microbiome plays a key role also in the pathogenesis of HIV/AIDS-and age-related comorbidities, like cardiovascular disease, stroke, malignancy, long-bone fractures, and renal impairment/dysfunction (Sortino et al., 2019). Moreover, the gut microbiota has effects on HIV drug metabolism, pharmacokinetics, and effectiveness (Imahashi et al., 2021).

THE GUT MICROBIOME AND HIV PRE-EXPOSURE PROPHYLAXIS
Few studies have investigated the impact of HIV PrEP on the gut microbiome, by conducting metagenomic analyses and next-generation sequencing (NGS) of the bacterial 16S/16S-23S ribosomal RNA (rRNA) gene(s) in highly adherent PrEP users to assess the relative abundance of family and genus and potential changes/shifts in the enteric microbiome. Features of these studies are summarized in Table 1. Dubé et al. (2018) were the first to detect and report alterations in the microbiome composition, with Erysipelotrichaceae (in particular, Catenibacterium mitsuokai, Holdemanella biformis, and Turicibacter sanguinis) and Streptococcaceae (especially, Streptococcus agalactiae, Streptococcus oralis, and Streptococcus mitis) being increased [by a mean log 2 (fold change) of 2.1, from 0.79 to 3.3%] and being decreased [by a mean log 2 (fold change) of −3.3, from 12.0 to 1.2%], respectively, already after 48 weeks of PrEP administration. No correlation with PrEP duration, antiretroviral levels, race, age, drug use, or abuse could be found. The authors hypothesized that changes in the gut microbiome may be linked with serious long-term side events, like colorectal malignancy, metabolic, and cardiovascular disease. However, at the moment, this remains a hypothesis not supported by clinical data, that, anyway, warrants further investigations, according to the same authors. Different findings were reported by Fulcher et al. (2019), who performed a cross-sectional study, comparing off versus on PrEP users, matched using propensity score 1:1 matching and performing logistic regression based on several covariates. These included age, race/ethnicity, BMI, smoking, alcohol and drug use (methamphetamine, and marijuana), and antibiotic use in the past month (single-dose azithromycin, ceftriaxone, or doxycycline), and sexual practices (recent receptive anal intercourse in the last 7 days and number of acts in the last month). Differently from Dubé et al. (2018) and Fulcher et al. (2019) found that the use of PrEP significantly correlated with an increase in Streptococcus. Other pathogens the abundance of which was increased were Mitsuokella, and Fusobacterium, while a decrease in Escherichia and Shigella abundance was observed. Of note, increased Fusobacterium was associated with increasing exposure to tenofovir, again differently from Dubé et al. (2018), who did not observe any correlation with antiretroviral concentrations. Similarly to Dubé et al. (2018) also Fulcher et al. (2019) warned about the potential long-term side-effects of oral PrEP: indeed, in the literature, increased Fusobacterium has been associated with inflammatory conditions, IBD, and colorectal cancer.
Finally, Perler et al. (2021) compared on and off PrEP individuals. In the group of PrEP users, the authors detected a statistically significant decrease in Pasteurellaceae, Corynebacteriaceae, Clostridiales Family XI (at the family level), and Finegoldia and Corynebacterium 1 (at the genus level), and an increase in Catenibacterium and Prevotella 2, while no changes were observed at the phylum level. Noteworthy, the increase in Catenibacterium had been previously reported by Dubé et al. (2018). Prevotella 9 was slightly higher in the PrEP user's group, but not in a statistically significant way. Moreover, this group had a higher number of male receptive anal sex partners, which correlated with the relative abundance of Prevotella 2, but not of Finegoldia or Streptococcus. No changes/shifts in Streptococcaceae and Erysipelotrichaceae could be observed, differently from previously published studies (Dubé et al., 2018;Fulcher et al., 2019). Of note, no differences could be detected in terms of human behaviors and lifestyles, like drugs/antibiotics exposure, sexual practices (anilingus), hygiene (rectal douching/enema), use of stool softeners, probiotic supplementations, use of lubricants, or use of saliva as a lubricant, and gastrointestinal symptoms, among others. Interestingly, sexually transmitted infections (STIs) were not found to impact the gut microbiome composition. Finally, alpha diversity did not change (as in previous studies; Dubé et al., 2018;Fulcher et al., 2019), differently from beta diversity. In line with the other studies overviewed in the present review (Dubé et al., 2018;Fulcher et al., 2019), the authors concluded that increased pathogens may lead to long-term periodontal disease, RA, insulin resistance, morbid obesity and metabolic impairments, and cardiovascular disease.
While all the studies analyzed consistently report changes and shifts in the gut microbiota, there are differences at the phylum, family, and/or genus level. This could be due to: (i) the different study design (cross-sectional versus longitudinal, case-control versus RCT), (ii) the sample size employed, (iii) the microbiome analysis methods and techniques used for specimen collection (rectal swab versus mucosal biopsy; Araújo-Pérez et al., 2012), and bioinformatics/statistical analysis, and (iv) the period of treatment (from a few months to some years, short-versus long-term). Moreover, further information should be collected about lifestyles, including dietary intake, exercise/physical activity, and sexual practices, which are known to modulate the gut microbiome composition. For example, the use of lubricants could have an effect on gut flora. Haaland et al. (2018) investigated whether the rectal application of a hyperosmolar lubricant impacted tenofovir drug tissue concentration in the rectum and related secretions and the composition of the gut microbiome. The authors found no effects on mucosal PrEP concentration but detected an impact of the use of lubricant on the gut microbiota, with decreased Bacteroides, increased concentrations of Prevotella (statistically borderline significant), and increased microbial diversity. A statistically significant interaction between the use of PrEP and lubricant was found for the relative abundance of the genus Alicyclobacillus. Of note, not all the studies analyzed, have adjusted for confounding factors.

CONCLUSION AND FUTURE PROSPECTS
As stated by Hughes et al. (2020), PrEP represents a unique opportunity to study the effects of Truvada ® and Descovy ® for PrEP on the gut microbiota without the interference of HIV, other drugs, and/or HIV/AIDS-related co-morbidities, as well as other confounding factors. These drugs are generally considered effective and safe. However, the study by Hughes and collaborators (Hughes et al., 2020) and the studies retained in the current review (Araújo-Pérez et al., 2012;Haaland et al., 2018;Hughes et al., 2020) have shown that they may alter and impair the gut microbiota, and repress the transcription of several nuclear transcription factors, inhibit the anti-inflammatory function of mucosal epithelial cells, by favoring a pro-inflammatory milieu, and promote gene signatures related to increased cell viability and proliferation as well as increase the abundance of pathogenic microbes, potentially linked to serious conditions and diseases. However, more data are urgently needed, especially concerning the long-term side effects of PrEP: despite the fact that the studies included in the present overview are a high-quality RCT, and two well-designed cross-sectional studies, evidence related to the impact of HIV PrEP on the gut microbiome in MSMs is still scarce and based on small populations. A better understanding of the complex, nonlinear interactions between the gut microbiota, sexual orientation/identity, and HIV prevention is expected to significantly improve PrEP adherence and potentially devise interventional strategies to counteract PrEP-related side effects.